I. Field of the Invention
This invention relates to the field of immunoassays, more particularly to reagents and methods useful for the rapid and sensitive quantification of the peptide hormone hBNP in a biological fluid such as plasma or serum.
II. Description of the Prior Art
BNP is a cardiac derived peptide hormone that circulates in the blood and exerts potent cardiovascular and renal actions. BNP is structurally similar to two other cardiac peptides, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). Porcine BNP was isolated from pig brain (Sudoh el al., Nature, 332:78-81, 1988) and, hence, was given the name xe2x80x9cbrain natriuretic peptidexe2x80x9d. In man the cardiac ventricle is the primary site of BNP synthesis. The sequence of human BNP (hBNP) was originally determined by the isolation and characterization of DNA clones from human genomic libraries (U.S. Pat. No. 5,114,923). hBNP is synthesized, in vivo, as a 108 amino acid precursor that is enzymatically cleaved to yield the mature hBNP peptide. Mature hBNP consists of a 32 amino acid peptide containing a 17 amino acid ring structure formed by two disulfide bonds (see FIG. 1).
Elevated expression of ventricular hBNP mRNA has been reported in congestive heart failure patients as compared to normal controls. Consistent with the increase in ventricular mass and expression of BNP mRNA, hBNP levels are elevated in patients with congestive heart failure and appear to correlate with disease severity. Plasma hBNP is also believed to provide a valuable predictive marker for heart disease. Elevated plasma hBNP has been reported in heart disease, following acute myocardial infarction and during symptomless or subclinical ventricular dysfunction (Mukoyama et al., J. Clin. Invest., 87:11402-1412, 1991) Motwani et al., Lancet, 341:1109-1113, 1993) (Yoshibayashi et al., New Eng. J. Med., 327:434, 1992). Reports from two major therapeutic trials, the SAVE trial (New Eng. J. Med., 327:669-677, 1992) and the SOLVD trial (New Eng. J. Med., 327:685-691, 1992) suggested that diagnosis and appropriate treatment of patients with asymptomatic left ventricular dysfunction could significantly reduce the incidence of fatal and non-fatal cardiovascular events and related hospitalizations.
For use in a clinical laboratory setting, it would be highly desirable to provide a diagnostic assay for hBNP which is sufficiently sensitive to measure clinically relevant titers of hBNP, sufficiently simple that it can be automated, requires a minimum amount of time to complete and preferably does not require the use of reagents having limited shelf lives. Normal levels of hBNP in plasma are quite low, on the order of 1 to 20 pg/mL. Typically, radioimmunoassays are used to measure titers in this range, although the use of radioactive reagents requires special handling, adds steps to the assay and involves the use of material having limited shelf life. A radioimmunoassay is commercially available for measuring hBNP, however, in addition to requiring the use of radioactive reagents, it is complex and cumbersome, requiring an extraction step, precipitation and several centrifugation steps, and takes several days to complete. European Patent Application No. 0 542 255 describes a radioimmunoassay for hBNP.
The present invention provides reagents and methods for the rapid and direct quantification of hBNP levels in biological fluids. The reagents and methods provided herein allow for the detection of hBNP titers at clinically relevant levels without the use of radioactive labelling (although radioactive labelling may be used, if desired). Moreover, they provide for the direct quantification of hBNP in plasma without the need for cumbersome extraction steps. The reagents and methods provided herein lend themselves readily to the automated systems used to conduct blood tests on a large scale in commercial clinical laboratories.
In one embodiment of the invention, there are provided monospecific antibodies to selected peptide epitopes within the hBNP molecule. Preferably, these monospecific antibodies are monoclonal antibodies. The monospecific antibodies of the invention are selected from the group consisting of:
(a) an antibody that is monospecific to a peptide epitope comprising amino acids 5-13 of hBNP;
(b) an antibody that is monospecific to a peptide epitope comprising amino acids 1-10 of hBNP; and
(c) an antibody that is monospecific to a peptide epitope comprising amino acids 15-25 of hBNP,
or functionally active fragments thereof.
A preferred antibody of the invention is a monoclonal antibody that recognizes and binds the peptide epitope comprising amino acids 5-13 of hBNP.
In another embodiment of the invention, there is provided a method for quantifying the amount of hBNP in a biological fluid using the reagents of the invention in a sandwich type immunoassay. The immunoassay may employ any of numerous labelling techniques to label and quantify immune complexes, with enzymatic labelling being preferred. This method of the invention comprises the steps of:
(a) contacting a sample of the biological fluid with a first antibody selected from the group consisting of:
(i) an antibody that is monospecific for a peptide epitope comprising amino acids 5-13 of hBNP;
(ii) an antibody that is monospecific for a peptide epitope comprising amino acids 1-10 of hBNP;
(iii) an antibody that is monospecific for a peptide epitope comprising amino acids 15-25 of hBNP;
(iv) an antibody that is monospecific for a peptide epitope comprising amino acids 27-32 of hBNP; and
functionally active fragments of (i)-(iv),
xe2x80x83and a second antibody selected from the group consisting of:
(i) an antibody that is monospecific for a peptide epitope comprising amino acids 5-13 of hBNP;
(ii) antibody that is monospecific for a peptide epitope comprising amino acids 1-10 of hBNP;
(iii) an antibody that is monospecific for a peptide epitope comprising amino acids 15-25 of hBNP;
(iv) an antibody that is monospecific for a peptide epitope comprising amino acids 27-32 of hBNP;
(v) a high affinity polyclonal antibody to hBNP; and functionally active fragments of (i)-(v),
xe2x80x83under conditions which allow the formation of a first antibody-hBNP-second antibody complex, provided that if the first antibody is monospecific to hBNP 27-32, then the second antibody is not a polyclonal antibody;
(b) binding a quantifiable label to said second antibody, prior to, simultaneously with or after formation of the first antibody-hBNP-second antibody complex; and
(c) determining the amount of hBNP in the sample by quantifying the label in the first antibody-hBNP-second antibody complex.
In other embodiments of the invention, there are provided methods for quantifying the amount of hBNP in a biological fluid using the reagents of the invention in competition type immunoassays. One such assay comprises the steps of:
(a) contacting a sample of the biological fluid with:
(i) an antibody that is monospecific for a peptide epitope comprising amino acids 5-13 of hBNP or a functionally active fragment thereof; and
(ii) hBNP or a fragment thereof comprising amino acids 5-13 of hBNP, having bound thereto a quantifiable label,
xe2x80x83under conditions that allow the formation of an antibody-hBNP complex; and
(b) determining the amount of hBNP in the sample by quantifying the label in the antibody-hBNP complex.
Another embodiment of the assay of the invention in a competition type format comprises the steps of:
(a) contacting a sample of the biological fluid with:
(i) an antibody that is monospecific for a peptide epitope comprising amino acids 1-10 of hBNP or a functionally active fragment thereof; and
(ii) hBNP or a fragment thereof comprising amino acids 1-10 of hBNP, having bound thereto a quantifiable label,
xe2x80x83under conditions that allow the formation of an antibody-hBNP complex; and
(b) determining the amount of hBNP in the sample by quantifying the label in the antibody-hBNP complex.
Another embodiment of the assay of the invention in a competition type format comprises the steps of:
(a) contacting a sample of the biological fluid with:
(i) an antibody that is monospecific for a peptide epitope comprising amino acids 15-25 of hBNP or a functionally active fragment thereof; and
(ii) hBNP or a fagment thereof comprising amino acids 15-25 of hBNP, having bound thereto a quantifiable label,
xe2x80x83under conditions that allow the formation of an antibody-hBNP complex; and
(b) determining the amount of hBNP in the sample by quantifying the label in the antibody-hBNP complex.
In the sandwich assays and the competition assays of the invention, the first antibody-hBNP-second antibody complex (in the case of a sandwich assay) or the antibody-hBNP complex (in the case of a competition assay) will usually be separated from the remainder of the biological fluid sample prior to quantifying the label in the complex. There are, however, known labelling techniques which allow for the direct measurement of labelled complex in the sample, i.e. without separating the complex from the sample, and such methods are considered to be within the scope of the invention. As merely exemplary of such methods, one can mention the scintillation proximity assay (Udenfriend, S. et al., Anal. Biochem., 161:494-500, 1987) and the assay described in Mathis, G., Clin. Chem., 41:1391-1397, 1995.
There are also provided by this invention fragments of hBNP which can be used as reagents in a competition type assay of the invention.
In one embodiment, the hBNP fragment of the invention has the formula
X1-V-Q-G-S-G-C-F-G-R-X2(SEQ ID NO:2),xe2x80x83xe2x80x83(I)
wherein X1 is selected from the group consisting of
hydrogen,
M-,
K-M-,
P-K-M- or
S-P-K-M- (positions 1-4 of SEQ ID NO:1)
and X2 is selected from the group consisting of
hydroxyl,
-K,
-K-M,
-K-M-D; and
-K-M-D-R (positions 14-17 of SEQ ID NO:1).
In another embodiment, the hBNP fragment of the invention has the formula
S-P-K-M-V-Q-G-S-G-C-X3(SEQ ID NO:3),xe2x80x83xe2x80x83(II)
wherein X3 is selected from the group consisting of
hydroxyl,
-F and
-F-G.
In another embodiment, the hBNP fragment of the invention has the formula
X4-M-D-R-I-S-S-S-S-G-L-G-X5(SEQ ID NO:4),xe2x80x83xe2x80x83(III)
wherein X4 is selected from the group consisting of
hydrogen,
K-,
R-K- and
G-R-K-
and X5 is selected from the group consisting of
hydroxyl,
-C,
-C-K,
-C-K-V,
-C-K-V-L (positions 26-29 of SEQ ID NO:1),
-C-K-V-L-R (positions 26-30 of SEQ ID NO:1),
-C-K-V-L-R-R and (positions 26-31 of SEQ ID NO:1)
-C-K-V-L-R-R-H (positions of 26-32 of SEQ ID NO:1).